All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Cancer remains among the leading causes of death in the United States and around the world. Various forms of cancer are differentially treated, depending in part on the location of a tumor. One particularly difficult group of tumors to treat includes those that reside in and near the brain. Treatment of brain tumors presents a number of problems, not the least of which being the dangers inherent in any surgical procedure involving regions of the brain and the tissue located nearby. There is little room for error and the consequences of even a minor surgical mishap can be devastating to a patient; brain damage, or even death may result. Still, where possible, surgery remains the preferred method of treatment for most brain tumors and is often performed in conjunction with radiation therapy and chemotherapy. However, even commonly referenced medical authority suggests that patients with brain tumors be referred to centers specializing in investigative therapies; an indication that conventional modes of treatment are not overwhelmingly successful.
Glioblastoma multiforme and anaplastic astrocytomas are classified in the category of brain tumors commonly known as malignant gliomas. Although not particularly common tumors themselves, they represent a class of tumors associated with significant rates of mortality and morbidity. Indeed, brain tumors are the third-most frequent cause of cancer-related death in middle-aged males and the leading cause of cancer death in children. According to the National Brain Tumor Foundation, approximately 190,000 people are diagnosed with primary or metastatic brain tumors in the United States each year. According to the Society for Neuroscience, approximately 20,000 cases of glioma are diagnosed each year, and more than half die within 18 months. For patients with the most severe, aggressive form of glioblastoma multiforme (“glioma” or “GBM”), median survival is less than a year. Current treatment for malignant glioma consists of surgical resection followed by radiation therapy and chemotherapy. However, this treatment generally fails in substantially changing the outcome for a patient. Thus, there remains a significant need in the art for improved methods for the treatment of cancer, and, in particular, for brain tumors.
Cancers are primarily comprised of a heterogeneous population of cells with marked differences in their proliferative potential. Cancer stem cells are a minor population of tumor cells that possess the stem cell property of self-renewal, and it is believed that dysregulation of stem cell self-renewal is a likely requirement for the development of cancer. (Al-Hajj, M. et al., Therapeutic Implications of Cancer Stem Cells, CURR. OPIN GENETIC DEV. 2004 14:43-47.)
Stem cells have the capacity to replicate themselves into cells with similar properties in order to maintain a pool of precursor cells. Adult stem cells, also called tissue stem cells, are found in differentiated tissues in which, in a controlled manner, they differentiate and/or divide to produce all the specialized cell types of the tissue from which they originate. Adult stem cells are often multipotent, capable to produce several but limited numbers of cell types. Normal tissue stem cells are typically defined by three common properties: (i) the presence of an extensive capacity for self-renewal that allows maintenance of the undifferentiated stem cell pool over the lifetime of the host; (ii) strict regulation of stem-cell number; and (iii) the ability to undergo a broad range of differentiation events to clonally reconstitute all of the functional elements within the tissue. Importantly, the stem cells in each tissue differ with respect to their intrinsic ability to both self-renew and to differentiate into particular mature cell types (Bixby, S. et al., Cell-Intrinsic Differences Between Stem Cells From Different Regions Of The Peripheral Nervous System Regulate The Generation Of Neural Diversity, NEURON 2002, 35:643-656).
The use of 6-bromoindirubin-3′-oxime (“BIO”) to maintain embryonic stem cells in an undifferentiated state is known in the art. (Sato et al., Maintenance of Pluripotency in Human and Mouse Embryonic Stem Cells through Activation of Wnt Signaling by a Pharmacological GSK-3-specific Inhibitor, NAT. MED. (2004), 10(1), pp. 55-63, and Meijer et al., GSK-3-selective Inhibitors Derived from Tyrian Purple Indirubins, CHEM. BIOL. (2003), 10(12), pp. 1255-1266.) Indirubin-type compounds are also used to inhibit GSK-3 and other protein kinases. (International PCT Pat. App. Pub. No. WO 2005/041954, filed Oct. 28, 2004.) BIO has been isolated, included pharmaceutically acceptable compositions, salts, or vehicles thereof, and methods of inhibiting GSK-3 activity with BIO in vitro or in a cell have been described.
Several genes initially linked to carcinogenesis have been implicated in the regulation of the normal stem-cell self-renewal process, including the Bmi-1, Notch, Wnt and Sonic hedgehog pathways. (Spink, K. E. et al., Structural Basis of the Axinadenomatous Polyposis Coli Interaction, EMBO J. 2000, 19:2270-2279; Taipale, J. et al., The Hedgehog and Wnt Signaling Pathways In Cancer, NATURE 2001, 411:349-354; Bhardwaj, G. et al., Sonic Hedgehog Induces the Proliferation of Primitive Human Hematopoietic Cells Via Bmp Regulation, NAT IMMUNOL 2001, 2:172-180; Austin, T. W. et al., A Role for the Wnt Gene Family In Hematopoiesis: Expansion of Multilineage Progenitor Cells, BLOOD 1997, 89:3624-3635.) For example, Reya et al. (A Role for Wnt Signaling in Self-Renewal of Hematopoietic Stem Cells, NATURE 2003, 423:409-414) demonstrated the dependence of normal HSC self-renewal decisions on Wnt-signaling through the canonical pathway. Willert et al. demonstrated the ability of purified Wnt3a to permit the in vitro expansion of transplantable HSCs. (Willert, K. et al., Wnt Proteins Are Lipid-Modified and Can Act as Stem Cell Growth Factors, NATURE 2003, 423:448-452.) Additional studies implicate the Wnt/b-catenin pathway in the maintenance of stem-cell self-renewal in other tissues as well. (Andl, T. et al., Wnt Signals Are Required for the Initiation of Hair Follicle Development, DEV CELL 2002, 2:643-653; Jamora, C. et al., Links Between Signal Transduction, Transcription and Adhesion in Epithelial Bud Development, NATURE 2003, 422:317-322.)
Cyclins and cyclin-dependent kinases (CDK) are key regulators in mammalian cell cycle. Regulation of these complexes occurs through cyclin production and destruction, relocation, inhibitory and activating phosphorylation events, relocation and also via the effects of other proteins. Each cyclin associates with one or two CDKs and most CDKs associate with one or two cyclins. CDK1 forms a complex with cyclin A/B and regulates phosphorylation of cytoskeleton proteins involved in mitosis. CDK2 and CDK3 form complexes with cyclin E which regulate the G1-S phase transition while the CDK2/CycA complex regulates S phase progression. CDK4/CycD and CDK6/CycD are activated by mitogenic signaling during early G1 and progressively accumulate as cells transition through this phase of the cell cycle. CDK5 is activated in postmitotic neurons and regulates neuron migration during brain development. CDK7/CycH is believed to be a link between transcription and cell cycle. CDK8/CycC and CDK9/CycT are involved in transcription. The kinase activity of CDKs is tightly regulated by phosphorylation and protein-protein interactions. Activation of CDKs requires binding to a specific cyclin and phosphorylation of a conserved threonine residue in a region called the T loop. Examining the phosphorylation of peptides by CDK/cyclin complexes suggests that both CDKs and cyclins play a role in recognizing substrates. A consensus sequence, (S/T)PX(R/K), is identified in the peptides that are phosphorylated by CDK/cyclins.
Cyclin-dependent kinase activity is regulated by T-loop phosphorylation, by the abundance of their cyclin partners and by association with CDK inhibitors of the Cip/Kip or INK family of proteins. The inactive ternary complex of CDK4/cyclin D and p27 Kip1/Cip1 requires extracellular mitogenic stimuli for the release and degradation of p27, which affects progression through the restriction point and pRb-dependent entry into S-phase. The active complex of CDK4/cyclin D targets the retinoblastoma protein for phosphorylation, allowing the release of E2F transcription factors that activate G1/S-phase gene expression. In HeLa cells, upon UV irradiation, upregulation of p16 INK4a association with CDK4/cyclin D3 leads to a G2 delay, implicating CDK4/cyclinD3 activity in progression through G2-phase of the cell cycle.